Endonuclease III
Introduction Every human cell experiences about 20,000 DNA damaging events per day due to reactive oxygen species (ROS). ROS, (generated from aerobic cellular metabolism, UV, etc.) can oxidize bases, create abasic sites, or cause single strand breaks. One mechanism to repair damaged DNA is the base excision repair pathway (BER). BER consists of four steps: recognition and removal of the lesion, cleavage of the remaining aldehyde, addition of the proper base, and finally the ligation of the phosphodiester bond. Endonuclease III (Nth) is one of many DNA glycosylases responsible for detecting and cleaving oxidized bases 2. Nth has two domains: an iron sulfur cluster and a glycosylase domain. The N-terminus of the human ortholog, NTH-like (hNTHL1), is predicted to be highly disordered and is not conserved in non-mammalian species. The structure and function of this region is still unknown, although it has been suggested that it aids in dimerization of hNTHL1 and may interact with DNA 4. Expression There is a truncated version of hNTH1 where the first 55 amino acids are excluded hNTH delta 55. This truncation removes a disordered region of the protein. Recombinant DNA techniques are used to over express the protein for experimental purposes. The hNTH delta 55 DNA is cloned into the pET-30a(+) expression vector. p-ET30a(+) is a 5422 base pair commercial vector. There are many restriction sites in the multiple coding sequence making cloning into the vector simple. There also a handful of common restriction enzymes that do not cut pET-30a(+) allowing for more advanced cloning techniques. Some key features of pET-30 are the T7 promoter and terminator sequences, the lacI operon, kanamycin coding sequence, and the N-term His tag and optional C-term His tag. The T7 sequences make it easy to sequence the vector to check for proper insertion of your plasmid DNA. The lacI operator is how expression is controlled. The kanamycin coding sequence selects for only completely ligated colonies. The His-tags aid in purification and the option to have them on either end allows for it to not interfere with the proteins activity. (The plasmid map highlights these features) In the case of hNTH delta 55 the N-term His-tag is used. The plasmid is transformed into the commercially available Rosetta2 p-Lyss e. coli cells. These cells contain the rare codon sequences (selected for with cloramphenicol) to allow for the expression of human proteins. The protein is expressed using a technique called autoinduction 5. This technique uses a glucose and glycerol solution as food. As the e. coli ''use the glucose and glycerol they produce lactose. When the supplied sugar solution is depleted the cells then use the lactose as food. This initiates the expression of your recombinant gene because of the lacI operon. The cells are grown at 20C for 60 hours. Purification The fresh cell pellet is resuspended in ice cold buffer with a protease inhibitor, PMSF. The is lysed by sonication and centrifuged to remove cellular debris (cell walls, DNA, etc.). The supernatant conaining the protein is purified by affinty chromotography, Ni-NTA. The His tag binds to the Ni beads and is out competed by imidazole for removal of hNTHL1 from the resin. The iron sulfur cluster has a yellow-brown color therefore samples that are yellow-brown are flown over a cation exchanger to further purify the construct. An S-column is a cation exchanger, NTH delta 63 has a predicted isoelectric point of 8.78, therefore it has a slight positive charge at pH 8. The colored fractions are pooled and this time concentrated to a volume of 500ul. Size exclusion chromatography is performed, using a superdex 200, to purify the protein to homogeneity. The colored fractions are analyzed using gel electrophoresis to assess purity. Uses Human NTHL1 is used in ''in vitro experiments to learn more about the enzyme. The truncated construct is used in X-ray crystallography experiments. Determining the structure of the enzyme will aid in our understanding of mechanism and function. Along with the crystallography experiments biochemical assays are performed to assess activity and DNA binding to different biological lesions. These constructs can easily be modified to known cancer variants. By assessing the activity and behavior of cancer variants we can learn more about how the enzyme is potentially important features of the enzyme. References 1 Katcher, H.L. and Wallace, S.S. Biochemistry 22 '(1983), 4071. PMID:6351916 2 Odell, I.D.''et al, ''Mol. Cell. Biol. '''31 '(2011), 4623. PMID:2193079 3 Liu X, and Roy R. J Mol. Biol. '321 '(2002), 265. PMID:12144783 4 Liu X, Choudhury S, and Roy R. J Biol. Chem. '''278 (2003), 50061. PMID:14522981 5 Studier, F.W. Protein Expression and Purification. '41 '(2005), 207. PMID:15915565